1. Field of the Invention
It is known that viable microorganisms, which are proteinaceous in nature, have extensive commercial uses. Microorganisms, including bacteria, yeast and fungi are capable of use in widely divergent fields. U.S. Pat. No. 3,438,863 discloses the use of Aspergillus niger, A. clavatus, A. wentii, Pencillium citrinum and P. luteum for citric acid production. U.S. Pat. No. 3,265,586 discloes bacterial alpha amylase enzymes derived from strains of Bacillus subtilis. Lactic-acid producing bacteria such as Streptococcus lactis, S. cremoris and S. diacetilactis are disclosed in U.S. Pat. No. 3,483,087. The production of galactose oxidase is disclosed in U.S. Pat. No. 3,186,921. The commercial production of ethanol from various yeast strains, e.g. Saccharomyces, is well known in the art. Because of an interest in decreasing consumption of petroleum products, the use of ethanol in internal combustion engines is receiving increased attention.
In many uses of viable microorganisms, the reaction is conducted in a reactor using a "batch" process. A large capital investment is required for the batch reactor equipment, in addition to the large space requirements and the amounts of energy required.
At present, citric acid, for example, is produced in large batch reactors which have a capacity of as much as 30,000 gallons. A fermentation substrate is prepared with suitable nutrients in a reactor, inoculated with citric acid-producing strains of A. niger and allowed to ferment. During fermentation, the reactor contents are continuously stirred. After fermentation, the contents of the reactor are removed, the mycelium filtered and citric acid recovered.
In the traditional "batch" method to make ethanol, a fermentable substrate is put into a reactor and inoculated with yeast. Here agin, during fermentation the contents are agitated. After fermentation, the reactor contents are removed, the mycelium separated and the alcohol solution recovered.
It is a goal in numerous applications involving viable microorganism to develop processes involving a fixed bed, where the microorganism is immobilized, a substrate is passed through the bed, and the product produced by the microorganism recovered. A successful fixed bed system would reduce the costs referred to above and reduce effluent problems by allowing reuse of the microorganisms.
Thus, it is a goal of research in citric acid production to develop processes involving a fixed bed, where the A. niger or other suitable cells are immobilized, a fermentation substrate is passed through the bed, and citric acid recovered. It is also a goal in ethanol production to develop processes involving a fixed bed, where the yeast cells are immobilized, the feedstock flows in continuously at one end of the bed, and the alcohol-containing mixture flows out the other end of the bed.
One method of immobilizing such microorganisms is by encapsulating the microorganisms within the gel lattice of a polymer. While this method of immobilizing microorganisms has an advantage in that the reaction conditions utilized to accomplish the entrapment are sufficiently mild so that often there is little alteration or deactivation of the microorganisms, it also had disadvantages in that the gel produced has poor mechanical strength. Low mechanical strength can produce compacting of the gel containing the microorganisms, which can cause plugging of the reaction system.
2. Prior Art
In Enzyme Microb. Technol., Vol. 1, 95-99 (1979), immobilization techniques using kappa-carrageenan for immobilization of Escherichia coli are described. Gel-inducing agents, such as various metal ions, amines and ammonium ions, are suggested. Also described is stabilization of the activity of the microorganisms by treating the immobilized cells with glutaraldehyde; also described is hardening the gelled material with a mixture of glutaraldehyde and hexamethylenediamine and a salt such as KCl. In J. Solid-Phase. Biochem., Vol. 2, 225-236, (1978) various methods for immobilizing glucose isomerase-producing microbial cells in kappa-carrageenan with gel-inducing ions, e.g. K+, NH.sub.4 +, and Ca.sup.2+, are described. The immobilized cells were treated with glutaraldehyde; glutaraldehyde and gelatin; and glutaraldehyde and hexamethylenediamine to improve the characteristics of the gel. The authors indicate that when the k-carrageenan immobilized cells were treated with hexamethylenediamine and glutaraldehyde, glucose isomerase activity was increased.
U.S. Pat. No. 4,138,292 is directed to immobilizing catalytically active substances and microorganisms. The patentee enumerates approximately two dozen catalytically-active microorganisms which can be immobilized. The patentee's procedure involves treating k-carrageenan with ammonium ion, a metal ion, a water-soluble organic amine or a water-miscible organic solvent. Approximately 50 organic amines are listed as being suitable for use as a gelling agent.
In column 7, the patentee suggests that the immobilized catalytically active substance may be further treated with a gel-hardening agent; approximately two dozen compounds are listed as being operable, including ammonia or an alkylenediamine and followed by reaction with an aliphatic dialdehyde, e.g., glutaraldehyde.